High pressure delivery system and method for treating pelvic disorder using large molecule therapeutics

ABSTRACT

The invention relates generally to methods and systems for the treatment of pelvic tissues using a high pressure injection device to deliver a therapeutic composition that includes a large molecule therapeutic agent, such as a nucleic acid or a polypeptide, to a target tissue in the pelvic area. Methods of the invention can improve delivery of the therapeutic agent into the tissue, which can be beneficial for the treatment of a pelvic tissue disorder, such as bladder and prostate tumors.

PRIORITY

This application is a divisional of application Ser. No. 13/328,674,filed Dec. 16, 2011, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/423,651, filed Dec. 16, 2010, entitled GENETHERAPY DELIVERY SYSTEM AND METHOD OF GENE TREATMENT, the disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to surgical tools and methods and, moreparticularly, to methods and devices for treating tissue in the pelvicarea using needleless or high-pressure jet injection devices forinjecting fluid into tissue.

BACKGROUND OF THE INVENTION

Pelvic health is an increasingly important health issue, e.g., based onan aging population. Treatment of pelvic conditions, such as urinarycontinence is an area of much investigation. Such conditions are desiredto be treated with therapeutic agents as opposed to invasive surgicalprocedures, when possible, in order to avoid risks of surgicalprocedures, such as infection, and recovery period and pain for thepatient. Therapeutic agents should be delivered with minimizeddiscomfort and procedure time, and with the best degree of accuracy ofdelivery location and delivery volume as possible. As such, there existscontinuing need to provide improved treatment methods and devices fordelivering therapeutic fluids to the pelvic region of a patient.

One type of disease of the pelvic tissue is bladder cancer, which is thesecond most common urological malignancy. Most bladder tumors are notinvasive into the bladder muscle layer (also known as superficialtumors). Non-invasive bladder cancer is commonly treated using surgicaltransurethral resection of the bladder tumor to take out the lesion.Recurrence of non-invasive bladder cancer is about 50% and depends oncertain prognostic factors such as tumor grade, tumor stage, and in situcarcinoma. Intravesical chemotherapy following surgical resection of thebladder tumor is often carrier out to delay or prevent recurrence orprogression to muscle-invasive bladder cancer. Despite surgicalresection and intravesical therapy, in a considerable percent ofpatients superficial lesions will recur, with some recurring lesionsprogressing to a higher grade or stage that form invasive cancers. Localinvasive cancer of the bladder is typically treated by radicalcystectomy, but this can result in the loss of pelvic floor functionsand adversely affect the patient quality of life.

Prostate disease is another pelvic floor disease that poses significanthealth risks for males. Diseases of the prostate include prostatitis,benign prostatic hyperplasia (BPH, also known as benign prostatichypertrophy), prostatic intraepithelial neoplasia (PIN), and prostaticcarcinoma.

SUMMARY OF THE INVENTION

The invention relates generally to systems and methods for treating apatient having a pelvic floor disease using a high-pressure injectiondevice to deliver a therapeutic composition that includes a largemolecule therapeutic agent useful for treating the disease.

In one aspect, the invention provides a method for treating orpreventing a disease of the pelvic floor. The method comprises a step ofdelivering a composition to a tissue selected from bladder, prostrate,urethral, kidney, vaginal, and rectal tissues, where the composition isdelivered to the tissue using a pressure in the range of 100 psi to 4000psi. The composition includes a therapeutic agent selected from thegroup consisting of nucleic acids and polypeptides and the therapeuticagent treats or prevents the disease in the tissue.

In another aspect, the invention provides a system for treating orpreventing a disease of the pelvic floor. The system comprises ahigh-pressure delivery device that comprises a pressure source and afluid compartment that can house a therapeutic composition. The deviceis capable of delivering the composition to the tissue with a pressurein the range of 100 psi to 4000 psi. The composition includes atherapeutic agent selected from the group consisting of nucleic acidsand polypeptides. In the system, the composition can be pre-leaded inthe device, or can be added to a fluid compartment of the device priorto delivering the composition.

In some modes of practice, the method and system can provide genetherapy which delivers DNA to cells to treat diseases based on geneticmutation, to alter the expression of particular proteins, or to causeapoptosis. Specific genes can be transcribed and then translated intoproteins, or the nucleic acid can prevent gene expression by antisenseor an interference RNA pathway. In one application, naked DNA isdelivered into tissues of the pelvic floor to treat or prevent pelvicflood condition. In another application, nucleic acid is included in aviral particle, a nanoparticle, or both, and delivered into tissues ofthe pelvic floor to treat or prevent pelvic flood condition. Genes andproteins could be delivered for other purposes, such as increasing orenhancing angiogenesis, wound healing, smooth muscle relaxation, or tocorrect a genetic mutation. One delivery means includes jet injection,which offers increased gene expression relative to needle injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an injection catheter system in accordance withembodiments of the present invention.

DETAILED DESCRIPTION

The invention relates to treatment methods and devices useful fordelivering (e.g., injecting or instilling) a large molecule therapeuticagent to tissue at or near target pelvic structures, such as the pelvicfloor. Large molecule therapeutic agents are represented by those suchas polypeptides, including proteins, and nucleic acids, such as RNA andDNA, and can include specific genes that can be transcribed and thentranslated into proteins.

In one example, naked DNA is delivered into tissues of the pelvic floorto address one of a variety of pelvic health issues. Genes (andtherefore proteins) could be delivered for many other purposes,including but not limited to, increasing or enhancing angiogenesis,wound healing, and smooth muscle relaxation, to correct a geneticmutation or to cause apoptosis. One delivery means includes jetinjection, which advantageously offers increased gene expressionrelative to needle injection. However, needle injection, including microneedles, is included within the scope of the invention.

The invention relates generally to surgical tools and methods and, moreparticularly, to methods and devices for treating tissue of the urinarytract (e.g., prostate tissue, kidneys, ureters, urethral tissue,bladder, etc.) of a subject, using needleless or high-pressure jetinjection devices for injecting fluid into tissue. The terms “patient”and “subject” are used herein to refer to mammals that can receivetreatment using the high-pressure injection device and associatedmethods. The mammal treated can be a human subject or a non-humansubject, such as domesticated pets, farm animals, and experimentalanimals, including, but not limited to dogs, cats, rabbits, rats, mice,sheep, horses, and cows.

Administration of the compositions using the high pressure injectiondevice can prevent or delay the onset of the symptoms of a disease (orcondition or disorder) of the pelvic tissue, can be used to alleviate orameliorate symptoms, or halt further progression of the disease. Successin treating disease (or condition or disorder) of the pelvic tissue canbe assessed by objective or subjective parameters, including imaging ofthe treatment site, biochemical or histological analysis of blood ortissue, and examination by a physician.

In some modes of practice the high-pressure jet injection device injectsa fluid composition that includes a nucleic acid which becomesincorporated into cells of the particular pelvic tissue that wastargeted. The nucleic acid is incorporated into, or “taken up” by thecells, and, in some cases, the cells biochemical machinery express theincorporated nucleic acid. Cells that have incorporated the nucleic acidcan be referred to herein as “transformed” or “transgenic” by the virtueof the nucleic acid having been incorporated into the cells.

A “nucleic acid,” as used herein can be any polynucleotide molecule ofcovalently-bonded nucleotide monomers, i.e., a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, whichcan include analogs of natural nucleotides. The nucleic acid can includethose that are natural, synthetically-prepared, modified (e.g., anucleic acid derivative), and enzymatically-treated. Exemplary nucleicacids include, but are not limited to, DNA and RNA, modified DNA andRNA, antisense oligonucleotides, antisense iRNA (immune ribonucleicacid), ribozymes, siRNA (small/short interfering RNA), and shRNA(small/short hairpin RNA). The nucleic acid can be a part of a plasmid,a phage, a cosmid, and episome, or an integratable DNA fragment. Thenucleic acid used to treat the pelvic floor condition can be isolatedfrom a variety of sources, genetically engineered, amplified, and/orexpressed/generated recombinantly, or chemically synthesized in vitro.

Antisense RNA and DNA are single-stranded polynucleotides (e.g,deoxyribonucleotide or ribonucleotide polymers) which can becomplimentary to a messenger RNA (mRNA) strand. Antisensepolynucleotides can be introduced into a cell and base pair to mRNAthereby physically obstructing the translation complex and inhibitingtranslation. One or more particular genes can be targeted for antisenseinhibition which results in decreased expression of the gene(s) whichcan be beneficial for the treatment of a disease associated with pelvictissue. The antisense oligonucleotide can be complementary to andcapable of hybridizing to a target nucleic acid. The antisenseoligonucleotide can have a length of less than 100 bases, such as about10 to about 50, or about 15 to about 40, bases in length.

The methods of the invention can be used to affect a process of RNAinterference in cells of the target tissue, which involves RNA-dependentgene silencing. An RNA molecule, such as short double-stranded RNA, isintroduced into the cytoplasm, where it interacts with proteins to forman RNA-induced silencing complex (RISC). After integration into theRISC, the interfering RNA base-pairs to its target mRNA and inducescleavage, which prevents the mRNA from being used as a translationtemplate. siRNA is short (generally about 20-25 nucleotides in length)double-stranded RNA which can be used to interfere with expression of aspecific gene via the RNA interference (RNAi) pathway. shRNA includes aRNA sequence and has a structure that includes hairpin turn that cansilence gene expression also via the RNA interference (RNAi) pathway. AshRNA approach may include use of stRNA (small temporal RNA) (Paddison(2002) Genes Dev. 16, 948-958).

In some arrangements, the nucleic acid can be present on a vector orexpression cassette from which the gene can be expressed after it isintroduced into the cell. The vector or expression cassette can includea promoter operably linked with the gene sequence that is desired to beexpressed. The promoter can be a tissue specific promoter for expressionin a target pelvic tissue, such as the promoters for the uroplakinsgenes UP1a and UP2 which can provide bladder tissue-specific expression.Optionally the vector or expression cassette can include othersequences, such as transcription termination signals or enhancershelpful in effecting expression. A nucleic acid can be considered to beoperably linked to another nucleic acid when the sequences are placedinto a functional relationship with each other, such as a promoteroperably linked to a coding sequence which affects the transcription ofthe sequence. The vector can be capable of transporting the desirednucleic acid and any other sequence to which it can be operably linked.Plasmid vectors are generally circular double stranded DNA loops intowhich DNA segments can be ligated. In viral vectors DNA segments can beligated into the viral genome. Some vectors (e.g., non-episomalmammalian vectors) can be integrated into the genome of a host cell uponintroduction into the host cell, and thereby are replicated along withthe host genome.

Exemplary vectors include viral vectors, plasmids, phages, phagemids,cosmids, and bacteriophages. Exemplary viral vectors include adenovirusvectors, retroviral vectors, and adeno-associated viral vectors. In somecases the cloning vehicle can comprise a plasmid or a bacteriophageP1-derived vector (PAC).

A nucleic acid can be administered to a target pelvic tissue using thehigh pressure injection device as “naked DNA” (see, e.g., U.S. Pat. No.5,580,859) or in the form of an expression vector, e.g., a recombinantvirus. Vectors administered in vivo can be derived from viral genomes,including recombinantly-modified enveloped or non-enveloped DNA and RNAviruses, such as from baculoviridiae, parvoviridiae, picornoviridiae,herpesviridiae, poxviridae, adenoviridiae, or picornaviridiae. Chimericvectors can also be employed which exploit advantageous merits of eachof the parent vector properties (See e.g., Feng, Nature Biotechnology15:866-870, 1997). Such viral genomes can be modified by recombinant DNAtechniques to include the nucleic acids for treating a pelvic floordisease, including those described herein; and can be further engineeredto be replication deficient, conditionally replicating or replicationcompetent. In alternative aspects, vectors are derived from theadenoviral (e.g., replication incompetent vectors derived from the humanadenovirus genome, see, e.g., U.S. Pat. Nos. 6,096,718; 6,110,458;6,113,913; 5,631,236), adeno-associated viral, or retroviral genomes.Retroviral vectors can include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), or human immuno deficiency virus (HIV); see, e.g., U.S. Pat. Nos.6,117,681; 6,107,478; 5,658,775; 5,449,614; Buchscher, J. Virol.66:2731-2739, 1992; Johann, J. Virol. 66:1635-1640, 1992).

In some aspects of the invention, the therapeutic composition includes apolypeptide. A polypeptide refers to an oligomer or polymer of two ormore amino acid residues linked by amide (peptide) bond(s). Polypeptidesencompass compounds referred to in the art as proteins, oligopeptides,peptides, and the like. Peptides are short polypeptides and typicallycharacterized by containing less than 50 monomer units. Proteins aremade from one or more polypeptides and are typically folded tofacilitate a biological function. Specific classes of polypeptidesinclude, but are not limited to, enzymatic polypeptides (enzymes),antibodies, antibody fragments, neuropeptides, and peptide hormones.Polypeptides can include common, naturally-occurring amino acidsresidues, including rare naturally-occurring amino acids, non-naturalamino acids, or combinations thereof. Incorporation of non-natural aminoacids into a polypeptide may serve to increase the stability, reactivityand/or solubility of the polypeptide. Polypeptides can also includethose that are modified with, or conjugated to, a biomolecule that isnot a polypeptide. For example, the polypeptide can be apolysaccharide-peptide conjugates (e.g., glyosylated polypeptides;glycoproteins), a poly(ethyleneglycol)-polypeptide conjugate (PEG-ylatedpolypeptides), or a peptide-nucleic acid (PNA) conjugate.

The system and methods of the invention using the high pressureinjection devices can be used to treat various types of bladder diseasesincluding bladder cancer. Urothelial carcinoma (transitional cellcarcinoma) is the most common type of bladder cancer (greater than 90%)in which the normal cells forming the innermost lining of the bladderwall undergo uncontrolled cell growth. Squamous cell carcinomas,representing 3%-8% of the bladder cancers, typically result from chronicbladder irritation or inflammation and originate from the thin, flatcells. Adenocarcinomas of the bladder are the least common (1%-2%).Transitional cells normally line the rest of the urinary tract includingthe kidneys, the ureters, the bladder, and the urethral and urothelialcarcinoma can develop in any of these areas.

Bladder cancers can be classified based on the extent of invasion,either superficial or invasive, into the bladder wall, which has severallayers. In superficial bladder cancer, characteristic of urothelial cellcarcinomas, the mucosa and lamina propria are only affected, which arethe innermost linings of the bladder. In invasive bladder cancers,characteristic of adenocarcinomas and squamous cell carcinomas, thecancerous growth has at least penetrated the muscular layer of thebladder wall. Bladder cancers are also typically characterized as lowgrade which are less aggressive growths, or high grade which are moreaggressive growths and display abnormal, phenotypically changed cells.Urothelial carcinomas can be in the form of papillary tumors which growas finger-like projections into the lumen of the bladder. Cancerousgrowth in the bladder can also develop in the form of a flat, red(erythematous) patch on the mucosal surface, referred to ascarcinoma-in-situ (CIS).

In some modes of practice a nucleic acid-containing composition includesone or more nucleic acid types which are used to treat tumor orcancerous tissue, and is delivered to the area of diseased tissue usinghigh pressure injection. Many approaches for treating tumor or canceroustissue involve using the administered nucleic acid to cause suppressionof the function of activated oncogenes, to initiate tumorself-destruction, or to restore the expression of functional tumorsuppressor genes. Exemplary targets of gene therapy for the treatment ofbladder cancer include the p53 tumor suppressor, transcription factorsZEB1 and ZEB2 involved in the epithelial-mesenchymal transition (EMT),epidermal growth factor receptor (EGFR)-related pathways, and genesinvolved in anti-angiogenic pathways.

Viral vectors can be used to deliver a desired nucleic acid. Viralvectors such as the adeno-associated virus (AAV) vector have beenreported to be non-pathogenic and offer long-term transgene expressionin vivo, and can include a desired nucleic acid for therapy of thetarget pelvic tissue. For example, the suppression effect ofAAV-mediated HSV-TK/GCV system on bladder cancer cells and in micexenograft models of bladder cancer has been investigated, and suggeststhat a rAAV-HSV-TK system can control tumour cell growth and providestrong anti-tumour efficacy in vivo. (Pan, J. G., et al. (2011) MedOncol. 2011).

In some modes of practice, the methods of the invention use of thenucleic acid composition to initiate tumour self-destruction through anapoptotic cell pathway (i.e., “suicide gene therapy”) for treatingdiseases of the pelvic system that are associated with unregulatedcellular growth. An exemplary therapeutic nucleic acid encodes encodesherpes simplex virus thymidine kinase (HSV-TK), which, when expressed ina target cell can phosphorylate ganciclovir (GCV) and be used to killthe cells. Phosphorylated GCV can interact with cellular DNA polymeraseand interfere with the synthesis of DNA resulting in the death ofdividing cells of the tumor tissue (Pan, J. G., et al. (2011) Med Oncol.2011). To further illustrate this type of nucleic acid technology,bifidobacterium infantis-mediated thymidine kinase (BI-TK) gene therapywas used in a rat model of bladder tumors by transformation of pGEX-TK(Tang W, et al. (2009) J Exp Clin Cancer Res. 28:155). After pGEX-TK wasadministered, ganciclovir (GCV) was intraperitoneally injected.Apoptosis was mostly notable in the BI-TK/GCV treatment group which hadthe highest level of caspase3 protein expression, resulting ininhibition of rat bladder tumor growth.

Another exemplary therapeutic nucleic acid encodes the Tumor necrosisfactor (TNF)-related apoptosis inducing ligand (TRAIL), which is amember of the TNF superfamily of cytokines and induces apoptosis in avariety of cancer cells, with minimal toxicity to normal cells. Toillustrate this type of nucleic acid technology, introduction of thehuman TRAIL gene into TRAIL-sensitive tumor cells using an adenoviralvector has been shown to result in the rapid production and expressionof TRAIL protein, followed by the death of the tumor cells (J. Immunol.(2000) 165:2886-94). Apoptosis was evidenced by certain cellular eventsincluding the activation of caspase-8 and the binding of annexin.

In some modes of practice, the methods of the invention targetinhibitors of apoptosis, such as members of the inhibitor of apoptosisprotein (IAP) family, with a nucleic acid for the treatment of bladdertumors. (IAPs) are highly conserved, are characterized by 70 amino acidbaculoviral repeats (BIR), and have wide spread roles in anti-apoptoticfunctions. Livin is an IAP family member that can inhibit apoptosis byinhibiting an inhibitor of IAPs. Livin expression has been reported invarious tumor cells and its over-expression of this gene appears to becancer cell specific. To illustrate this type of technology, in onestudy phosphorathioate modifying was used to synthesize antisenseoligonucleotides targeting Livin, followed by transfection into humanbladder cancer cell 5637 (Liu, C., et al. (2010) J Exp Clin Cancer Res.29:63). Livin antisense nucleotide effectively inhibited Livinexpression and tumor cell proliferation through enhanced caspase3activity and apoptosis of tumor cells. In a nude mouse transplantabletumor model, Livin expression was inhibited and caspase3 expression wasincreased resulting in enhanced apoptosis and slowed tumor growth.

Survivin is an anti-apoptotic protein expressed in many cancerousgrowths. Survivin inhibits apoptotic enzymes, and its overexpressionleads to uncontrolled cancer cell growth. Downregulation of survivinexpression has been targeted as a way to sensitize tumor cells topromote cell apoptosis. To illustrate this type of technology, in onestudy small interfering RNA (siRNA) targeting survivin was transfectedinto T24 cells using a liposome approach, which resulted in a decreasein survivin protein (Ku, J. H., et al. (2010) BJU Int. 106:1812-6). Thiscorrelated with a decrease in cell growth, G2/M arrest, and an increasein the fraction of cells undergoing apoptosis. The inhibition ofsurvivin expression increased caspase-3 activity in T24 cells, which ledto apoptosis. As such, in some modes of practice the high pressureinjection method uses siRNA to target survivin for the treatment ofbladder cancer.

As another example, anti-apoptotic proteins of the Bcl-2 family, such asbcl-2 and bcl-xL can be targets of gene therapy for the treatment ofbladder cancer. To illustrate this type of technology, in one study thecytotoxic effects of bcl-2 and bcl-xL antisense-oligodeoxy-nucleotides(AS-ODNs) in benign urothelial and transitional cell carcinoma (TCC)cell lines were investigated (Gabriel, U. (2008) Oncol Rep. 20:1419-23).Bcl-2 AS-ODNs showed higher cell mortality in TCC cells, whereas toxiceffects on normal urothelium was minor.

The invention also contemplates delivery of a composition containing anucleic acid that results in increased expression of cytokines usefulfor the treatment of pelvic diseases. For example, the nucleic acid canencode interleukin-15 (IL-15), which can stimulate the CD8(+) T-cell andnatural killer (NK) cell proliferation. To illustrate this type oftechnology, in one study intravesical liposomal IL-15 gene delivery andits antitumor effect were tested in a mouse orthotopic bladder cancermodel. (Matsumoto, K., et al. (2011) Hum Gene Ther. 22:1423-32), usingtransurethral delivery of liposome-mediated plasmids. Instillation withIL-15 gene plasmid showed inflammatory cells induced around the tumors,and infiltration of CD8(+) T cells around the tumor nest. After theintravesical IL-15 gene therapy, the growth of rechallenged subcutaneousMBT-2 cells in surviving mice was inhibited again via tumor-specificcytotoxic T lymphocytes.

Another cytokine useful for gene therapy for the treatment of bladdertumors is IL-23, in the sense that it can provide a tumor vaccine effectand can be used to combat tumorogenic growth. IL-23 plays a role in theinflammatory response against infection, and upregulates matrixmetalloprotease MMP9 expression, increases angiogenesis, and reducesCD8⁺ T-cell infiltration, and has been implicated in the development ofcancerous tumors. To illustrate this type of technology, in one study anexpression vector containing the IL-23 gene was introduced into mousebladder carcinoma (MBT2) cells, and mice vaccinated with the MBT2/IL-23cells inhibited the tumour growth of parental MBT2 cells injected at adistant site (Kuramoto, T., et al. (2010) BJU Int. 108:914-21). CD8⁺ Tcells were the main effector cells for the direct antitumour effect.

Another useful gene for providing protective anti-tumor immunity is theCD40 ligand (CD40L). CD40L is a member of the TNF superfamily andprimarily expressed on activated T cells. CD40L-CD40 interaction caninduce activation in an antigen-presenting cell (APC) in associationwith T cell receptor stimulation by MHC molecules on the APC. Toillustrate this type of technology, in the orthotopic mouse model ofbladder cancer, following intravesical instillation a SA-sCD40L fusionprotein was durably immobilized on the biotinylated mucosal surface ofbladder wall (Zhang, Z., et al. (2011) Acta Oncol. 50:1111-8). SA-sCD40Ltreatment prolonged the survival of MB49 tumor-bearing mice and cured50% of mice with MB49 superficial bladder. In another study, patientswith invasive bladder cancer scheduled for cystectomy or patients withT(a) tumors were treated with three cycles of intrabladder Clorpactin™WCS-90 (sodium oxychlorosene) prewash, followed by AdCD40L, which is anadenoviral vector expressing the CD40 ligand (AdCD40L) (Malmström, P.U., et al. (2010) Clin Cancer Res. 16:3279-87). Patients experienced noadverse events to the vector therapy, and gene transfer was detected inbiopsies, and bladders were heavily infiltrated with T cells. Histologicevaluation indicated that AdCD40L therapy reduced the load of malignantcells.

The invention also contemplates delivering agents via the high pressuredelivery system that provide control over gene products that causetranscriptional silencing by CpG island methylation of tumor suppressorgenes, and which can be used to treat cancerous growths. For example,depletion of the DNA methyltransferase DNMT1 in human bladder cancercells has been shown to induce demethylation and reactivation of thesilenced tumor-suppressor gene CDKN2A. To illustrate this type oftechnology, and in one study, selective depletion of DNMT1 using eitherantisense or siRNA resulted in lower cellular maintenancemethyltransferase activity, global and gene-specific demethylation andre-expression of tumor-suppressor genes in human cancer cells (Robert,M. F., et al. (2003) Nat Genet. 33:61-5). Specific depletion of DNMT1potentiated the ability of 5-aza-2′-deoxycytidine to reactivate silencedtumor-suppressor genes.

In some modes of practice, in addition to the high pressure delivery ofthe large molecular therapeutic according to methods of the invention,treatment can optionally include the administration of one or morechemotherapeutic agents. If desired, one or more chemotherapeutic agentscan be administered using any suitable route. In some cases, thechemotherapeutic agents can be administered intravesically, which can bedone using a standard catheter, or using the high pressure injectiondevice as described herein. Chemotherapeutic agents that have been usedto treat bladder cancers include N,N′N′-triethylenethiophosphoramide(ThioTEPA), adriamycin (doxorubicin, ADM), epirubicin (EPI), mitomycin C(MMC), valrubicin (AD32), pirarubicin (THP), gemcitabine, apaziquone(EOquin™), and Vicinium™ (anti-Ep-CAM humanized scFv-exotoxin A fusionprotein).

The invention also contemplates treating interstitial cystitis/painfulbladder syndrome (IC/PBS) with a large molecule therapeutic agent viathe high pressure delivery device. IC/PBS is a condition that manifestsitself in discomfort or pain in the bladder and surrounding pelvicregion. The cause of IC/PBS is unclear, but symptoms resemble those ofbacterial infection, without infection being present. To illustrate thistype of technology, one type of gene therapy to treat IC/PBS hasinvolved the targeted and localized expression of enkephalin in afferentnerves that innervate the bladder by gene transfer usingreplication-defective herpes simplex virus (HSV) vectors in a rat modelof bladder hyperactivity and pain (Yokoyama, H., et al. (2009) Hum GeneTher. 20:63-71). Replication-deficient HSV vectors encodingpreproenkephalin, which is a precursor for Met- and Leu-enkephalin wereinjected into the bladder wall of female rats. Functionally, enkephalinvector-treated animals showed reductions in bladder hyperactivity andnociceptive behavior induced by intravesical application of capsaicin.

Gene therapy for the treatment of a bladder disease can also beperformed by placing a gene of interest under the control of a promoterthat provides tissue-specific expression in bladder tissue. Theuroplakins (UP) are structural proteins and fundamental components ofurinary tract epithelium. Two uroplakins genes (UP1a and UP2) aretranscribed only in urothelium, and are therefore bladder-specificpromoters. Gene therapy viral vectors under the control of a UP promotercould be used to drive therapeutic gene expression in high-UP-expressingtransitional cell carcinoma. (Olsburgh, J., et al. (2003) J. Pathol.99:41-49) A uroplakin II promoter was used in the construction ofCG8840, a urothelium-specific adenovirus variant that eliminatedestablished bladder tumors in combination with docetaxel (Zhang, J., etal. (2002) Cancer Res. 62:3743-50).

Another target of gene therapy can be genes that modulate the assemblyof the cell's cytoskeleton. For example, gelsolin is regulator of actinfilament assembly and disassembly. Gelsolin is an actin regulatoryprotein that is undetectable or reduced in human bladder tumors comparedwith normal epithelial cells. To illustrate this type of technology, inone study recombinant adenovirus encoding wild-type gelsolin (Ad-GSN)was transduced into human bladder cancer cell lines KU-7 and UMUC-2(Sazawa, A., et al. (2002) J Urol. Sep;168:1182-7). KU-7 cells wereintroduced into the bladder of nude mice, followed by 3 injections intothe urethra with Ad-GSN. Bladder cancer cell growth was inhibited afterthese cells were transduced with Ad-GSN in vitro.

Another pelvic disease that can be treated using the high pressureinjection system of the current invention is kidney disease. Forexample, proteinuria (albuminuria) is a condition in which urinecontains an abnormal amount of protein, which is thought to result fromdamaged glomeruli of the kidney. As another example, diabeticnephropathy is a progressive disease where the capillaries in the kidneyglomeruli undergo angiopathy, and caused by diabetes mellitus. Asanother example, polycystic kidney disease (PKD) is a cystic geneticdisorder of the kidneys. MiRNA expression has been shown to differbetween the kidney and other organs as well as between different kidneyregions, and have been found to be functionally important in models ofPKD and diabetic nephropathy. The deletion of an endoribonuclease in theRNase III family called Dicer, which cleaves dsRNA and pre-miRNA intoshort siRNA, results in proteinuria and severe renal impairment in mice.In some modes of practice, modifying the expression of Dicer and/ormiRNAs involved in the kidney disease by injection of the therapeuticcomposition using the high pressure injection device is anothercontemplated method of treatment (Li, J. Y., (2010) Nephrology,15:599-608).

Chronic kidney disease (CKD) is also characterized by accumulation ofextracellular matrix. Myofibroblasts that are α-SMA-positive whichproduce large amounts of TGF-β1 are thought to play an important role inthe development of interstitial fibrosis. To illustrate this type oftechnology, in one study inhibition of fibrosis by hepatocyte growthfactor (HGF) was examined using HGF transgenic mice (HGF-Tg) withangiotensin II (Ang II) infusion (Iekushi, K., et al. (2010) JHypertens. 28:2454-61). HGF-Tg mice showed significantly decreased AngII-induced renal fibrosis and lesser numbers of interstitialmyofibroblasts. The antifibrotic action in HGF-Tg mice was concordantwith a decrease in TGF-β1, collagen type I and IV mRNA expression and anincrease in MMP-2 and MMP-9 expression. Furthermore, HGF-Tg mice treatedwith Ang II showed apoptosis of myofibroblasts. Inhibition of theFAK-ERK-MMP signaling cascade by specific inhibitor or siRNAsignificantly decreased HGF-induced myofibroblast apoptosis. One miRNA(miR-192) can also act as an effector of transforming growth factor-βactivity in the high-glucose environment of diabetic nephropathy (Li, J.Y., (2010) Nephrology, 15:599-608).

In another study, thrombospondin-2 (TSP-2) was investigated anendogenous regulator of matrix remodelling and inflammation in ananti-Thy1 glomerulonephritis model of experimental kidney disease(Daniel C, et al. (2009) Am J Physiol Renal Physiol. 297:F1299-309).TSP-2 overexpression inhibited both glomerular endothelial and mesangialcell proliferation, resulting in a reduced glomerular cell number andglomerular tuft area.

Autoimmune glomerulonephritis is associated with a significant immuneresponse with glomerular crescentic formation and fibrosis in thekidney. Overexpression of Smad7, which is a member of the SMAD family ofproteins, transduces extracellular signals from transforming growthfactor beta ligands to the nucleus where they activate downstream TGF-βgene transcription and attenuates both renal fibrosis and inflammationin rat remnant kidney. In another study, the Smad7 gene was transfectedinto the kidney and its overexpression blocked both renal fibrosis andinflammatory pathways (Ka, S. M., et al. (2007) J Am Soc Nephrol.18:1777-88).

Therefore, in another mode of practice, the therapeutic compositiondelivered by the high pressure injection device provides a therapeuticagent which increases HGF expression, decreases TGF-β1 expression, orinterferes with the FAK-ERK-MMP signaling cascade.

A nucleic acid-containing therapeutic compositions for treatment ofkidney diseases can be included in a nonviral, viral, or a cellularvector. For kidney tissue, one efficient vector for in vivo transfectionhas shown to be adenovirus. For example, glomeruli, blood vessels,interstitial cells, and pyelum can be transfected with high efficiencyusing an adenovirus that carries a nucleic acid of interest.

Recombinant human EPO (hEPO) has been successfully used to treat anemiaassociated with kidney disease using carried out for a considerableperiod of time. A genetic therapy approach for the production of hEPO isdesirable because of the disadvantages of administration of the hEPOprotein. To illustrate this type of technology, in one study anadenovector expressing EPO was constructed by inserting the human EPOcDNA was inserted into the pAd-lox shuttle vector containing thecytomegalovirus (CMV) promoter and simian virus-40 (SV40) polyA site(Lippin, Y., et al. (2005) Blood, 106:2280-2286). The adenovector wasused to create autologous genetically modified tissue that expressedhEPO.

In other modes of practice, the method of the invention is used for thetreatment of a prostate disease. For example, the treatment of prostatecancer can be performed by delivering a composition having a nucleicacid which alters the expression of an immunomodulatory gene, andapoptosis gene, a tumor suppressor gene, or an anti-oncogene in prostatetissue.

For example, gene therapy using the prostate-specific antigen (PSA)sequence can be used for treatment. PSA is a well known marker forprostate cancer the diagnosis and management. To illustrate this type oftechnology, in one study an enhancer/promoter construct 5′ end of thehuman PSA gene was inserted into adenovirus type 5 DNA to create aprostate-specific enhancer-containing virus (Rodriguez, R., et al.(1997) Cancer Res. 57:2559-63). The virus destroyed largeandrogen-sensitive human prostate adenocarcinoma (LNCaP) tumors andabolished PSA production in nu/nu mouse xenograft models with a singleintratumoral injection.

In another study, a functional DNA-lipid complex encoding theinterleukin 2 (IL-2) gene was administered to patients with locallyadvanced prostate cancer (CaP) (Belldegrun, A., et al. (2001) Hum GeneTher. 12:883-92). The IL-2 gene therapy was well tolerated, and therewas evidence of systemic immune activation, based on an increase in theintensity of T cell infiltration seen on immunohistochemical analysis oftissue samples from the injected tumor sites, and increasedproliferation rates of peripheral blood lymphocytes that were coculturedwith patient serum collected after treatment. A transient decreases inserum prostate-specific antigen (PSA) was seen in most patients.

The system and methods of the invention are used to treat a disease orcondition of present in at a location in the female genitourinary tract.In some modes of practice, the high pressure injection is used to treata fibroid, such as a uterine fibroid, or a cancerous growth inendometrial lining. Uterine fibroids are benign tumors that originatefrom the uterine myometrium (smooth muscle) and the accompanyingconnective tissue of the uterus. Fibroids include uterine leiomyomas,fibromyomas, and fibromas. For instance, a therapeutic compositionhaving can be injected into fibroids, or the uterine artery.

To illustrate this type of technology, in one study Eker rats withMRI-confirmed uterine leiomyomas were provided with direct intra-tumorinjection of adenovirus (Ad)-mediated delivery of herpes simplex virus 1thymidine kinase gene (HSV1TK) followed by ganciclovir (GCV) treatment(Hassan, M., et al. (2009) Gynecol Obstet Invest. 68:19-32). Theadenovirus treatment decreased uterine fibroid volume, increasedcaspase-3 activity, Bax expression, and TUNEL apoptosis marker, and itdecreased cyclin D1, PCNA, Bcl2, and PARP protein expressions.

The system and methods of the invention can also be used to treat adisease or condition of present in at a location in the lowergastrointestinal tract, such as the rectum or large intestine.

The therapeutic composition delivered via the high pressure deliverydevice can have a therapeutic agent (e.g., nucleic acid or protein) inan amount sufficient to provide a therapeutic effect in a patient. Theamount of the therapeutic agent provided to a patient can depend onvarious factors, such as the pharmacodynamics of the therapeutic agent,including the therapeutic agent potency, which is a measure of thequantity of therapeutic agent needed to produce a desired effect, thetherapeutic agent half life or stability, the ability of the therapeuticagent to reach its site of action (e.g., the ability of a nucleic acidto enter an intracellular space), the amount of tissue to be treated,the duration of treatment, and the like. The amount of therapeutic agentprovided to a patient can be described as a single dose, or as acumulative dose over a determined time period.

For example, a nucleic acid can be provided to a patient in an amountover a wide dosage range, such as from nanograms to milligrams. In manymodes of treatment, and amount of nucleic acid in the nanogram tomicrogram range is administered to the patient, such as from about 10nanograms to about 900 micrograms, about 100 nanograms to about 100micrograms, or about 500 nanograms to about 10 micrograms. A therapeuticpolypeptide can be administered to a patient in the same dose ranges.

Composition which can be delivered by the high pressure jet injectiondevices can include one or more excipient components to enhance deliveryof the nucleic acid into the cells, or to promote its integration and/orfunction in the cells. In some preparations the excipient material is anon-viral material that enhances nucleic acid delivery, by promotinguptake of the nucleic acid into the cell. Material having a positivecharge such as cationic lipids or polycations like polylysines andpolyethylenimines can condense nucleic acids into small particles.Examplary excipient materials include polyamidoamines, dendrimers,peptide carriers, lipopeptides, and lipopolymers. The charge density ofa polycation (the carrier) can operate to complex and the then cause therelease of the gene from the complex in the cytosol.

Specific examples include multivalent polycationic beta-cyclodextrinshaving a beta-cyclodextrin core oligoethyleneamine arms to facilitateDNA binding, encapsulation, and cellular uptake (Journal of the AmericanChemical Society (2008) 130:4618-4627); and polyethylene glycol(PEG)-polyethylenimine (PEI) conjugate polymers (U.S. Pat. No.7,060,498).

The composition can optionally include small solid particulates. Smallsolid particulates can be in the form of microparticles, microspheres,nanoparticles, or nanospheres, can include a large molecule therapeuticagent, and can be used to facilitate delivery of the therapeutic agentinto the pelvic tissue. The therapeutic agent present in the small solidparticulates can be released to provide a therapeutic effect after thehigh pressure injection. If such particles or other particles are mixedwith the liquid in the pressure chamber, these are also ejected duringthe delivery process and can behave in a manner similarly to the liquidsurrounding them.

The use of microparticles, microspheres, nanoparticles, or nanospherescan be beneficial in to protect large molecule therapeutic agent fromthe sheer forces associated with the ejection of material from the highpressure delivery device. In these cases, therapeutic agent within theparticulate, not directly on the particulate surface, is protected fromthese forces during delivery.

In some preparations, the small particulates include the therapeuticagent and an excipient agent used to form the particulates that is notreadily soluble in water, so they can be maintained in a particulatestate. In some preparations the small particulates include thetherapeutic agent and a biodegradable polymer. Exemplary biodegradablepolymers include polyesters such as polylactic acid,poly(lactide-co-glycolide), polycaprolactone, polyhydroxybutyrate,polyorthoesters, polyphosphazine, polymethylidenemalonate,polyalkeneanhydrides, polypeptides, polyanhydrides, and the like.

An exemplary jet injection device will now be discussed. The jetinjection device ejects a therapeutic “fluid” from a distal end of anelongate shaft inserted into or placed adjacent to the patient tissue.The devices can include multiple orifices that may be stationary ormoveable relative to a shaft of the device, for ejecting a pressurizedfluid at multiple locations. Embodiments that include multiple orificescan include an extended, expanded, or extendable chain, string, array,or sequence (e.g., “daisy chain”). Orifices may be located at anextension mechanism (“orifice extension”) such as extendable or fanningneedles or needleless fluid delivery orifices.

The pressurized fluid source can include a source of fluid and a sourceof pressure. The pressure source may be mechanical (such as a spring),pneumatic, hydraulic, electric, etc., as will be understood. Further,the pressure source may be mechanically or electronically controlled.The pressure source can cause a fluid contained in a fixed or variablevolume chamber to be pressurized to a transient pressure, at theinjection orifice, that is sufficiently high to allow the fluid to beejected from the injection orifice with sufficient force to penetrateinternal tissue such as to traverse the urethra and to then penetrate adesired distance into target pelvic tissue. An endoscope or otheroptical feature can be included for use with the injection device.

Various device structures, components, methods and techniques describedand depicted in U.S. Patent Publication No. 2006/0129125 andInternational Publication No. WO2007/079152 are envisioned for use,alone or in combination, with the present invention. As such, the entiredisclosures of the above-referenced publications are incorporated hereinby reference.

In one embodiment, such as that shown in FIG. 1, a jet injectioncatheter device or system 10 can include a proximal portion 12 and adistal portion 14, and a shaft or body portion 16 extendingtherebetween. The proximal portion 12 generally includes a handle 18,and a connection port or assembly adapted to interconnect with a fluidsource 36. The fluid source 36 is in operative and fluid communicationwith the proximal portion 12 via a conduit 34. The fluid source 36 caninclude a reservoir and pressure source capable of pressurizing andadvancing fluid contained in the source. The fluid source can begenerally “remote” (FIG. 1) from the proximal portion or the distalportion 14, or provided generally proximate or directly attached to thedevice components.

A working lumen or channel 17 extends within the shaft 16 and contains afluid delivery lumen 22 such that the lumen 22 is adapted to movelongitudinally along the length of the body 16 to allow the distal endof the fluid delivery lumen 22 to extend from the tip of the distalportion 14 as an orifice extension. The high-pressure injectate isdelivered to the target tissue from the fluid delivery lumen 22. Inparticular, the injectate traverses from the fluid source 36, into theworking channel 17, and out of the fluid delivery lumen 22. Shaft 16 caninclude a fiber optic feature 30, e.g., an endoscope device, having alight source 20 to transmit light to the distal portion 14. In oneembodiment, the fluid source 36 can include a fully-pneumatic source orinjection console. Such a console configuration promotes safety andfunctionality. Power for the system 10 and console can come from a CO₂tank in which pressure is monitor by a gauge.

A variety of materials may be used to form portions or components of thesystems 10, including nitinol, polymers, elastomers, fluid systems andcomponents, thermoplastic elastomers, metals, ceramics, circuitry,springs, wires, plastic tubing, and the like. The device 10, and itscomponents and methods may have a number of suitable configurationsknown to one of ordinary skill in the art.

A method for the delivery of the therapeutic agent can involve insertingand then moving shaft or body portion 16 of the device through one ormore parts of the urogenital tract of a male (urethra, bladder) orfemale (vagina, urethra, bladder, uterus, fallopian tubes), or the lowergastrointestinal tract (rectum, large intestine) to position the distalportion 14 end of the device at a desired treatment site. The device canthen be actuated to eject the liquid composition at high pressure fromthe distal end of the device into the tissue. Liquid composition can bedelivered in a single ejection of fluid, or more than one ejection offluid. The distal end of the device can be moved slightly betweeninjections to target a larger area of tissue.

A fluid stream or jet of composition ejected from an aperture ofapertures at the distal end of the device can be of a size (e.g.,diameter), velocity, pressure, and volume to penetrate into the tissuein a desired manner. The stream can be considered to be a relativelyhigh velocity, high pressure, small diameter jet that enters anddisperses within the tissue. The therapeutic composition can be carriedas a multi-directional collection of particles (e.g., a “cloud”) ordroplets within the plaque. The injection pressure refers to thestagnation pressure of the impinging jet, which is an important factorthat determines the penetration capability of the injected fluid. Thepressure p at the aperture or outlet is equivalent to the averagestagnation pressure across the outlet area. Stagnation pressure is thepressure exerted by moving fluid when forced to come to rest, forexample, by impingement on a target.

Exemplary pressures of a fluid at a pressure chamber can be at least 100pounds per square inch (psi) (˜0.7 megapascal (MPa)), for example, suchas in the range of about 140 psi (˜1 MPa) to about 4000 psi (˜27.5 MPa),and more specifically in the range of about 140 to about 2000 psi (˜13.8MPa) are contemplated for injecting the therapeutic composition intotissue of a target region of the pelvic area. Injection pressures fortreatment of pelvic tissues may be lower that what is typically used forepidermal needless injections (E.g, see Shergold, O. A., and Fleck, N.A. (2004). Proc. R. Soc. Lond. A., 460:3037-3058).

It is believed that that impact of the fluid stream or jet on the targettissue creates of a hole in the tissue through a cracking process. Theaccumulation of injected liquid slows down until it reaches a maximumpoint as the hole becomes deeper. Stagnation of the jet at the end ofthe hole expands the liquid into the tissue in a spherical pattern(Schramm-Baxter J, Mitragotri S (2005). J. Control. Rel., 106(3):361-373.).

The duration of injection of the fluid stream or jet of the therapeuticcomposition can be performed for a desired period of time. In somemethods of injecting, the duration of injection is controlled by one ormore feature(s) of the injection device, such as a solenoid or valve, inorder to meter the flow or stream of the composition from one or moreapertures at the distal end of the device. The injection can include oneor more high pressure “pulses” of the composition from one or moreapertures. The duration of a pulse can be short such as in the range ofabout 5 milliseconds to about 1 second, or more specifically in therange about 50 milliseconds to about 0.6 seconds.

Small quantities of liquid are delivered to target pelvic tissue usingthe high pressure injection device. Cubic millimeters of liquid can beejected from the distal end of the device under high pressure tofacilitate delivery of the therapeutic substance to pelvic tissue. Insome modes of practice a quantity in the range of about 0.1 cm³ to about2 cm³, or more specifically about 0.25 cm³ to about 1 cm³ can bedelivered to pelvic tissue per pulse of the high pressure injectiondevice. Treatment can include a series of short pulses to deliver adesired amount of composition to target tissue in the pelvic area.

In some modes of practice, the distal end of the device is moved betweenpulses to change the direction and/or location of the injectedtherapeutic composition. For example, in use the shaft or body portionof the device is moved internally in the body to position the distal endat different tissue locations between pulses. Movement can be carriedout in a proximal to distal direction, rotationally, or both. Thetreatment area can be targeted and very small, wherein only a singlepulse can provide the desired treatment, or the treatment area can belarger, wherein multiple pulses are performed after positioning thedistal end of the device at different locations in the body.

What is claimed is:
 1. A system for delivering a therapeutic agent topelvic tissue comprising (a) a high pressure delivery device comprisinga pressure source, a fluid compartment that can house a therapeuticcomposition, and a distal end configured for placement at a location inthe male or female urogenital tract or lower gastrointestinal tractselected from bladder, prostate, urethral, kidney, vaginal, andendometrial tissues, wherein said device is capable of delivering thecomposition from a distal end of the device at a pressure in the rangeof 100 psi to 4000 psi, and (b) a therapeutic composition comprising anucleic acid or a polypeptide for treating or preventing a disease ofthe pelvic tissue, wherein the nucleic acid or polypeptide is present ina nanoparticle or microparticle; said nanoparticle or microparticlecomprising an excipient polymeric material selected from the groupconsisting of polylactic acid, poly(lactide-co-glycolide),polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyphosphazine,polymethylidenemalonate, polyalkeneanhydrides, polypeptides, andpolyanhydrides.
 2. The system of claim 1 wherein the high pressuredelivery device is configured to deliver the composition to the tissueusing a pressure in the range of 140 psi to 2000 psi.
 3. The system ofclaim 1, wherein the high pressure delivery device is configured todeliver the composition having a volume in the range of 0.1 cm³ to 2cm³.
 4. The system of claim 3 wherein the volume is in the range 0.25cm³ to about 1 cm³.
 5. The system of claim 1 wherein the high-pressureinjection device comprises a distal portion comprising a shaftconfigured for insertion into the urethra of the subject.
 6. The systemof claim 1 wherein the high-pressure injection device comprises aneedleless distal end having one or more aperture(s) for the ejection ofthe composition.
 7. The system of claim 1 wherein the distal end isconfigured for placement at bladder tissue.
 8. The system of claim 1,wherein the nucleic acid promotes apoptosis of the cells of the tissuethe composition is injected into.
 9. The system of claim 1, wherein thenucleic acid promotes an RNA interference pathway to modulate expressionof a target gene.
 10. The system of claim 1, wherein the nucleic acid ispresent in a nanoparticle.
 11. The system of claim 1 wherein , Ofpreferred excipient polymers are selected from the group consisting ofpoly(lactide-co-glycolides), polyhydroxybutyrates, and polyorthoesters.